HIV-1 is the causative agent of AIDS. Three viral enzymes -- reverse transcriptase (RT), integrase (IN), and protease (PR) -- have essential roles in the replication of HIV-1. We are engaged in a long-term effort to study HIV-1 RT, with the expectation that this information will be useful in the development of more effective anti-RT drugs. Our strategy has involved the analysis of both wild-type and mutant HIV-1 RTs, including drug-resistant mutants. Some of this purified RT has been used by our long-term collaborator, Dr. Eddy Arnold (Rutgers University), for structural studies. We have used purified HIV-1 RT to study the biochemical properties of RT mutants, including drug-resistant mutants. There are two clinically important classes of inhibitors of HIV-1 RT: nucleoside analogs (NRTIs) and nonnucleoside inhibitors (NNRTIs). Both are used to treat HIV-1 infections;however, there are serious problems with drug toxicity and with the development of resistance. A major focus of our work on HIV-1 RT is the mechanism(s) of RT inhibitor resistance. a. RT mutants. We have been studying the effects of mutations in reverse transcriptase (RT) and in the HIV-1 genome on reverse transcription. We showed that a large percentage of mutations in the thumb subdomain make RT susceptible to PR cleavage;in some cases, this susceptibility creates a temperature-sensitive phenotype. We proposed that the mutations that lead to protease (PR) cleavage partially unfold RT, exposing sites where PR can cleave. We will make recombinant versions of some of the PR-sensitive RTs and examine whether they can be denatured at lower temperatures than WT. We will test additional RT mutations outside the thumb subdomain, and examine whether mutations in other HIV-1 proteins make these proteins more susceptible to PR cleavage. We are trying to obtain second-site revertants of RT mutants;so far, we have only obtained true revertants. b. Fidelity of HIV-1 replication in cell culture. We used a LacZalpha complementation assay similar to the assays used by the Pathak and Mansky labs to measure the fidelity of HIV-1 replication in vitro. We improved the efficiency of the assay, which has allowed us not only to measure the mutation rate, but also to determine the positions in LacZalpha where mutations frequently arise (hotspots). The data show that (1) all of the published in vitro assays using purified RT overestimate the in vivo error rate and fail to correctly identify the mutational hotspots;(2) HIV-1 replication is not more error prone than the replication of other retroviruses;(3) which strand of LacZalpha is in the RNA genome does not affect the overall error rate, or the types of errors made, but it does affect the hotspots;(4) based on preliminary data, mutations in RT affect which sites are mutational hotspots;(5) there is evidence, in our cell culture system, that ADAR may cause multiple mutations. We are analyzing a number of RT mutants using this system. c. NRTIs and NRTI resistance. We are continuing to investigate the mechanisms of resistance to nucleoside analogs (NRTIs), using both structural analysis and biochemical assays. We are also interested in elucidating the mechanism of action of compounds that do not cause an immediate/ complete block to DNA synthesis. Ongoing structural analysis in Dr. Arnold's group should shed light on the underlying mechanism(s). With the retirement of Dr. Victor Marquez (National Cancer Institute), we no longer have a source of new NRTIs. We are planning to test the toxicity (in mice) of a promising NRTI, D-carbaT. We have sufficient quantities of both D-carbaT and a more potent prodrug form for the toxicity testing. We are trying to select D-carbaT-resistant viruses in tissue culture. d. New NNRTIs. We are generating and testing new nonnucleoside inhibitors (NNRTIs). We have several promising compounds that are able to inhibit wild-type (WT) and several common NNRTI-resistant viruses with IC50s below 5 nM. These compounds have CC50s for cells in culture more than 4 logs higher than their IC50s. Additional compounds are being designed and synthesized based on the structures/properties of the most promising compounds, modeling, and X-ray structures (generated by Dr. Arnold's group) of the compounds in complexes with WT and drug-resistant RTs. e. RTCs and PICs. We have been working on the development of protocols to isolate reverse transcription complexes (RTCs) and preintegration complexes (PICs) from infected cells, to determine the host and viral proteins present in the RTCs and PICs. We have used the presence of viral DNA as a marker, and have used immunological approaches to enrich for the RTCs and PICs. Although progress has been made, we are not yet able to produce sufficient quantities of material pure enough to identify the proteins present in RTCs and PICs. [Corresponds to Hughes Project 1 in the April 2007 site visit report of the HIV Drug Resistance Program]